Non-tumorigenic cell lines for expression of genes

ABSTRACT

Non-tumorigenic cell lines capable of indefinite growth in serum-free media are disclosed. The cell lines are capable of expressing exogenously introduced genes, and may be derived from mouse embryo cells. Methods for producing proteins utilizing these cell lines, and methods for selectively controlling the growth of the cell lines are also disclosed.

This application is a continuation of U.S. patent Application Ser. No.08/177,637, filed Jan. 4, 1994, issued as U.S. Pat. No. 5,474,930, onDec. 12, 1995; which application is a continuation of U.S. patentApplication Ser. No. 07/852,490, filed Mar. 16, 1992, now abandoned;which application was a continuation of U.S. patent Application Ser. No.07/540,460, filed Jun. 18, 1990, now abandoned; which application was acontinuation of U.S. patent Application Ser. No. 07/035,814, filed Apr.8, 1987, now abandoned.

TECHNICAL FIELD

The present invention relates generally to cell lines capable ofexpressing selected genes of interest, and more particularly, to celllines that can be propagated indefinitely under serum-free conditions,can be reversibly growth-inhibited, and are capable of expressingexogenously introduced genes.

BACKGROUND ART

There have been numerous attempts to express selected genes of interestusing a variety of different cell lines. Conventional establishedmammalian cell lines, for instance, have been widely utilized in lightof their capacity to grow quickly to high cell densities and insuspension, both desirable characteristics for the production ofbiological products. Examples of commonly used cell lines in this regardinclude the BHK, CHO, arid COS cell lines. Often however, the growth ofestablished cell lines such as these cannot be sufficiently controlled.This is a significant disadvantage in situations in which a high celldensity has been achieved, and the primary purpose becomes collection ofthe biological product rather than generation of increasing cellnumbers. In addition, established cell lines almost without exceptionexhibit an abnormal karyotype. These cell lines are often eithertumorigenic in vivo, or give rise to tumorigenic cells upon furtherproliferation and extended culture. Tumorigenicity is a significantconcern when selecting a cell line from which biological products willbe isolated.

An additional disadvantage of conventional established cell lines isthat they are isolated and propagated in culture media containing aserum supplement. Further, these cell lines often require a serumsupplement for continued growth (Barnes and Sato, Cell 22:649-655,1980). This requirement for serum creates a source of additionalexpense, problems relating to quality control and reproducibility of theserum, and problems in isolating desired biological substances.

In contrast to established cell lines, normal cell lines in earlypassages exhibit a predominantly diploid karyotype and arenon-tumorigenic. However, these cells, known as primary cells,characteristically undergo alterations upon multiple passages.Consequently, the culture eventually degenerates or undergoes multiplegenetic and phenotypic changes, often resulting in the development oftumorigenic cells, making further passage of primary cells undesirablefor the production of biological products.

While there have been attempts to utilize serum-free media to generatecell lines capable of continuous growth without subsequent degenerationor chromosomal aberration, these efforts have been only partiallysuccessful. Consequently, there is a need in the art for a cell linecapable of indefinite growth under serum-free conditions, the cell linefurther being capable of expressing exogenously introduced genes. Inaddition, the cell line should be capable of growth to high density insuspension, the growth of the cell line also being subject to selectivecontrol. The present invention fulfills this need and further providesother related advantages.

DISCLOSURE OF THE INVENTION

Briefly stated, the present invention discloses a non-tumorigenic celllines capable of indefinite growth in serum-free media, the cell linesbeing further capable of expressing exogenously introduced genes. In oneparticular embodiment, the cell line is derived from mouse embryo cells.(Hereinafter referred to as "serum free mouse embryo" or "SFME").

Another aspect of the present invention discloses a method for producinga desired protein. The method generally comprises (a) introducing a geneencoding the desired protein into a non-tumorigenic cell line capable ofindefinite growth in serum-free media; (b) growing the cell line in aserum-free medium; and (c) isolating the protein product encoded by thegene and produced by the cell line.

Yet another aspect of the present invention discloses a method forselectively controlling the growth of a non-tumorigenic cell linecapable of indefinite growth in serum-free media. The method generallycomprises growing the cell line in serum-free media and subsequentlysupplementing the media, either briefly or continually, with aneffective amount of a selected biological substance. Suitable biologicalsubstances in this regard include serum, plasma, conditioned cellculture medium, tissue extracts, or purified or partially purifiedcomponents thereof, peptide growth factors, glycocorticoids, thyroidhormones, retinoids or other hormonal or related compounds.

Other aspects of the invention will become evident upon reference to thefollowing detailed description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth of a representative mouse embryo cell linederived in serum-free medium. , culture medium containing 10% calfserum; O, serum-free culture medium, as described in Example I, infra; ,serum-free culture medium containing 10% calf serum.

FIG. 2 depicts karyotyping data obtained with Swiss mouse and BALB/cmouse embryo cell lines grown in serum-containing and serum-free culturemedium.

(A) primary Swiss mouse embryo cells cultured in medium containing 10%calf serum (2 population doublings);

(B) post-crisis Swiss mouse embryo cells cultured in medium containing10% calf serum (20 population doublings);

(C) SFME cells cultured in serum-free medium (200 population doublings);

(D) BALB/c mouse embryo cells cultured in medium containing 10% calfserum (7 population doublings);

(E) BALB/c SFME cells cultured in serum-free medium (70 populationdoublings).

FIG. 3 shows the inhibitory effect of serum upon the growth of SFMEcells. O, serum-free culture medium, as described in Example I, infra;, culture medium containing 10% calf serum; , serum-free culture mediumcontaining 10% calf serum; □, serum-free culture medium lackingepidermal growth factor (EGF); Δ, 3-days growth in culture mediumcontaining 10% calf serum, followed by culture in serum-free medium.

FIG. 4 illustrates growth inhibition of SFME cells in serum-free mediumcontaining various concentrations of plasma or serum. , calf serum; O,plasma.

BEST MODE FOR CARRYING OUT THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

Serum-free media: Tissue culture media in which the mammalian bloodproduct supplement, usually serum, is replaced with a definedsupplement. The defined supplement contains discrete amounts of knowncomponents that, in combination, support the growth of cultured animalcells. A typical serum-free medium formulation may include nutrients,buffers, hormones, salts, antibiotics, proteins, growth factors, andenzymes.

Indefinite growth: A property of some cultured cells, especiallyestablished cell lines, that permits extended propagation of one lineageof cells. This characteristic is in contrast to that exhibited by mostnormal diploid cells isolated and cultivated in vitro, which undergosenescence upon multiple passages. Within the present invention,indefinite growth includes culture for more than thirty generations.

As described herein, Swiss SFME cells have been cultured for 200generations with no evidence of senescence, and BALB/c SFME cells havebeen cultured for 150 generations without senescence. As compared toSwiss or BALB/c embryo cultures maintained under conventional,serum-containing conditions, the SFME cell lines have been grown forten-fold or greater the number of generations, with no evidence of adecrease in growth rate.

Non-tumorigenic: Substances or cells that do hot give rise to tumorswhen injected in vivo. Tumorigenicity is generally a correlate ofphenotypic or genotypic changes that result in uncontrolled growth ofabnormal cells. Within the present invention nontumorigenic cells arethose cultures which can be injected subcutaneously into syngeneic orathymic mice at 10⁷ per mouse with no evidence of a decrease in growthrate.

As noted above, the present invention discloses non-tumorigenic celllines capable of indefinite growth in serum-free medium. An additionaladvantage of such cell lines, as compared to the cell lines described inthe prior art, is that the growth of the cell lines of the presentinvention can be controlled. Through control of growth, it is possibleto substantially inhibit increases in the cellular biomass, whileoptimizing production of the expressed biological product. A furtheradvantage of the disclosed cell lines is that purification of thedesired biological product from a non-tumorigenic cell eliminates therisk of co-purification of tumorigenic materials.

The present invention also allows the extended in vitro growth inserum-free medium of non-tumorigenic cells capable of expressing foreigngenes. A variety of mammals and tissues may be used for the derivationof cell lines suitable for use herein. Preferred cells in this regardare derived from mouse embryos and exhibit a predominately diploidkaryotype. Particularly preferred cells are derived from embryosobtained from Swiss and BALB/c mice. It will be evident to one skilledin the art that embryonic and non-embryonic tissue derived from avariety of mouse species, as well as other warm-blooded species,including human, may be utilized within the present invention.

The non-tumorigenic cells of the present invention may be used toproduce and recover biological products encoded by exogenouslyintroduced foreign genes. Preferred genes include those encoding tissueplasminogen activator, factor VIII, interleukin II, insulin, growthhormone, tumor necrosis factors, antibodies, superoxide dismutase andother enzymes, peptide hormones and hormone receptors, and interferons.However, it will be evident to one skilled in the art that a variety ofother foreign genes could be expressed by the disclosed non-tumorigeniccells. These foreign genes need only be properly introduced into thenon-tumorigenic cell line in an appropriate configuration andorientation to obtain production and recovery of desired biologicalproducts. The cell lines disclosed within the present invention may alsobe utilized in conjunction with a selection system using dominantmarkers (for instance, neomycin resistance). In addition, thenon-tumorigenic cell lines may be suitable host cells for anamplification system for introduced foreign genes.

As noted above, the non-tumorigenic cell lines described herein arecapable of indefinite growth in serum-free medium. In a preferredembodiment, the cells may be grown in culture medium containing adefined supplement. A preferred defined supplement includes insulin,transferrin, epidermal growth factor (EGF), high-density lipoprotein(HDL), and fibronectin. It will be evident to one skilled in the artthat this particular supplement is merely exemplary of those that may beutilized within the present invention. For instance, some components maybe substituted for others (e.g., insulin-like growth factors forinsulin; transforming growth factor alpha for epidermal growth factor;bovine serum albumin containing lipid for high-density lipoprotein;polylysine for fibronectin; and iron salts for transferin). Further,other factors might be added to the culture medium, such as tumorpromoters, additional hormones and/or growth factors, bovine serumalbumin, low concentrations of serum or plasma, or modified plasmapreparations with reduced inhibitory activity. Fibronectin might beeliminated from the culture medium formulation to obtainanchorage-independent growth of the present cell lines. Alteration ofculture medium components may also allow derivation of sublines of thenon-tumorigenic cell lines of the present invention or their equivalent.In addition, other supplements may be added to the medium formulation toenhance protein production from a particular foreign gene construct (forexample, addition of steroid hormones where the foreign gene is operablylinked to asteroid hormone-responsive promoter).

A method for producing a foreign gene product utilizing the cell linesof the present invention is also disclosed. In a preferred embodiment,foreign genes are introduced by transfection into the non-tumorigeniccells described herein. In this regard, dextran sulfate-, calciumphosphate-, and electrophoration-mediated transfaction are preferred,with calcium phosphate-mediated transfaction particularly preferred. Theforeign gene product is expressed by the non-tumorigenic cells, and thebiological product can then be purified by a variety of techniques wellknown in the literature. These techniques include immuno-affinitychromatography, high-performance liquid chromatography, ion-exchangechromatography, precipitation techniques, hydrophobic or saltinteraction techniques, isoelectric focusing, and electrophoresistechniques. In addition, similar techniques may be applied to theisolation of the foreign gene product from the culture medium in whichthe cells are grown or from the cells themselves.

A method for selectively controlling the growth of non-tumorigenic celllines capable of indefinite growth in serum-free medium is alsodescribed within the present invention. In a preferred embodiment,growth is controlled by supplementing the non-tumorigenic cell culturemedium with a selected amount of a synthetic or purified biologicalsubstance. Particularly preferred biological substances are serum,plasma, conditioned cell culture medium, tissue extracts, and purifiedor partially purified components thereof. However, it will be evident toone skilled in the art that a variety of blood products may be utilizedwithin this methodology. Such blood products may include plasmaproducts, such as Cohn fractions, or other blood product preparationsisolated by chromatographic, precipitation, affinity, or electrophoretictechniques. Hormones, steroid hormones, peptide hormones, nutritionalcomponents, and vitamins or related compounds, such as retinoids,present in the blood may be used, either as free components or bound tocarrier molecules. These free or bound preparations may then be added tothe non-tumorigenic cell culture medium in an amount sufficient tocontrol the growth of the cell line.

One advantageous use of the system described herein would be in theintroduction of a gene under the influence of a hormone-responsivecontrol element (e.g., the glucocorticoid responsive MMTV promoter) thatresponds to a hormone that inhibits SFME cell growth. Treatment of cellswith such a hormone would simultaneously inhibit all growth andstimulate protein synthesis.

To summarize the examples which follow, Example I describes a serum-freemedium useful for the isolation and propagation of a non-tumorigeniccell line. The derivation of the SFME mouse embryo cell lines in thisserum-free medium is also disclosed. Example II illustrates that thecells which are isolated and maintained in serum-free medium exhibit apredominately diploid karyotype, while cell lines which are isolated inserum-containing medium display an abnormal tetraploid karyotype.Example III illustrates that Swiss SFME mouse embryo cells, which werecultured for over 200 generations in serum-free medium, werenon-tumorigenic upon injection of the cells subcutaneously into 4- and7-week-old male and female athymic Swiss mice. The mice were observedfor six months, and no evidence of any tumorigenic activity wasobserved. However, oncogene-transformed SFME cells did show tumorigenicqualities after injections into athymic Swiss mice. Example IV setsforth an analysis of the effects of individual defined supplementcomponents in the growth of SFME cells in vitro. Example IV presents astudy of the inhibitory effects of the addition of serum to cellsisolated and grown in serum-free medium. Example VI describes thetransfection of the SFME cell line with the Ha-ras gene.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES EXAMPLE I

Serum-Free Medium for the Preparation and Maintenance of Mouse EmbryoCell Cultures

The serum-free medium used was a one-to-one mixture of Ham's F12 andDulbecco-modified Eagle's medium containing 4.5 g/l glucose (F12:DME)supplemented with bovine insulin (10 ug/ml; Sigma Chemical Co., St.Louis, Mo.) , human transferrin (25 ug/ml; Sigma) , human high-densitylipoprotein (HDL; 20 ug/ml; Meloy Laboratories, Springfield, Va.), mouseepidermal growth factor (EGF; 50 ng/ml; Collaborative Research, Inc.,Waltham, Mass.), human fibronectin (20 ug/ml; Meloy), sodium selenite(10 nM), human platelet-derived growth factor (PDGF; 1 unit/ml),penicillin (200 U/ml), streptomycin (200 ug/ml) ampicillin (25 ug/ml),15 mM 4-(2-hydroxy-ethyl)-1-piperazine-ethanesulfonic acid (pH 7.4), and1.2 g/ml sodium bicarbonate.

Sterile stock solutions of supplement components may be stored in therefrigerator, or may be stored in aliquots at -20° C. for longer periodsof time. Water utilized for the preparation of concentrated stocksolutions of supplement components and for the preparation of the mediumis purified by passage through a Milli-Q (Millipore, Bedford, Mass.)water purification system immediately prior to use. F12:DME is preparedfrom powdered formulations (GIBCO, Grand Island, N.Y.), and may bestored frozen at -20° C. for a maximum period of three weeks. Allsolutions and medium formulations are stored in reusable plasticcontainers. Polypropylene tubes are used for concentrated stocks, and250 ml polystyrene flasks are used for storing media. All pipets andculture vessels are disposable plastic (Falcon or Corning).

Insulin, transferrin, EGF and HDL are added to the F12:DME medium afterplating of the cells, by means of addition of small aliquots fromconcentrated stocks. Insulin is prepared at 1 mg/ml in 20 mM HCl;transferrin is made at 5 mg/ml in phosphate-buffered saline. Bothinsulin and transferrin concentrated stocks are filter-sterilized afterpreparation. EGF, PDGF and fibronectin are obtained as sterile,lyophilized powders from commercial sources (e.g., CollaborativeResearch; Meloy). These lyophilized preparations are reconstituted withsterile water or sterile buffered salt solutions. HDL is obtainedcommercially (Meloy) as a sterile solution.

Fibronectin is provided to the cultured cells by precoating cultureflasks for 30 minutes with 4 ml of a solution of fibronectin at 20 ug/mlin F12:DME, with removal of the precoating solution prior to plating thecells.

The SFME mouse embryo cell line was derived in serum-free medium bytrypsinization of minced, pooled 16-day Swiss mouse embryos, followed byplating of the cells at a density of 3×10⁶ cells/25 cm² tissue cultureflask. Trypsinization was accomplished with 0.1% crude trypsin and 1 mMethylenediaminetetraacetate (EDTA) in phosphate-buffered saline. Thetrypsinized cell suspension was then diluted into an equal volume ofserum-free culture medium containing 0.1% soybean trypsin inhibitorprior to centrifugation of the cells and resuspension in fresh medium.The cells were then grown in a 5% CO₂ -95% air atmosphere at 37° C.

Mouse embryo cell cultures were derived in serum-free medium from bothBALB/c and Swiss embryos. At high cell density, the cells exhibitedfibroblastic morphology, with spindle-shaped cells organized intocharacteristic swirls. Cells maintained in serum-free media grewexponentially without a significant time lag and without crisis (FIG.1). The number of cells in primary cultures six days after plating wasthree times higher in serum-free media than in serum- containing media.Cells were split approximately once per week, at a ratio of 1:4 to 1:10.The mouse embryo cells were cultured continually for 200 generations.

Serum-free medium containing the defined supplement of this Example willsupport the growth of some mouse embryo cell lines established inconventional serum-containing media. In addition, this serum-free mediumis related in nutritional and hormonal composition to other mediadeveloped for the growth of established lines of mouse embryo cells(Pipas et al., Cancer Cells 2:355-363, 1983; Chiang et al., In VitroCell Devel. Biol. 21:707-712, 1985).

EXAMPLE II

Karyotyping of the Swiss and BALB/c SFME Cell Lines

SFME Swiss and BALB/c cells carried for over 100 generations inserum-free medium were treated with colchicine and karyotyped. The cellswere predominately diploid (FIG. 2). No abnormal chromosomes wereidentified by Giemsa banding. Translocations were detected in someclones derived from the serum-free Swiss cultures, suggesting thatcloning or freezing procedures may affect karyotype. One clonecontaining a translocation was examined further, and found to remainnontumorigenic with a modal chromosome number of 40. Serum-free derivedcultures contained a small fraction of cells (4 to 7 percent) withkaryotypes in the tetraploid range.

Cell cultures which were grown in serum-containing media, with orwithout the addition of the described defined supplement, underwent awell-defined crisis, exhibiting abnormal karyotypes and tumorigenicqualities. After several population doublings, 100% of the metaphases ofSwiss SFME cells grown in serum- containing medium contained one or moretranslocated marker chromosomes (FIG. 2). No chromosomal abnormalitieswere identified in the serum-free derived Swiss and BALB/c SFMEcultures.

Chromosome assignments carried out on a number of metaphases revealedthat monosomies and trisomies did not involve the same chromosomes,indicating random loss and gain of chromosomes from metaphase tometaphase, rather than a particular chromosomal aberration. This degreeof division infidelity was also observed in primary cultures of mouseembryo cells in serum-containing medium that were processed and analyzedin an identical manner. The cells were confirmed to be of mouse originby isozyme and chromosome analysis, and were free of bacterial andfungal contamination as determined by examination of blood agar andbroth cultures inoculated with cells and medium from the cultures.

EXAMPLE III

Examination of the SFME Cell Lines for Tumorigenic Capacity

The tumorigenic capacity of the Swiss SFME line was analyzed byinjection of various numbers of cells into athymic mice. Athymic micewere used for these in vivo assays of tumorigenicity because SFME embryocultures were initiated from outbred mice. The tumorigenic capacity ofBALB/c SFME cells was analyzed by injection of cells into syngenicBALB/c mice. The results of in vivo assays are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        TUMORIGENIC PROPERTIES OF                                                     MOUSE EMBRYO CELL CULTURES                                                    Cells     Conditions        Results                                           ______________________________________                                        BALB/c    BALB/c mouse injection;                                                                         0/4 with tumors                                   SFME      10.sup.7 cells    after 120 days                                    Swiss     Athymic mouse injection;                                                                        0/8 with tumors                                   SFME      10.sup.7 cells    after 240 days                                    KBALB*    Athymic mouse injection;                                                                        4/4 with tumors**                                           10.sup.6 cells    after 14 days                                     RasSFME***                                                                              Athymic mouse injection;                                                                        4/4 with tumors                                             10.sup.6 cells    after 14 days                                     ______________________________________                                         *Kirsten sarcoma virustransformed 3T3 mouse embryo cells                      **1 cm diameter tumor                                                         ***Human Haras-transformed Swiss SFME cells selected for growth in            serumcontaining medium                                                   

The SFME cell lines are non-tumorigenic in athymic mice when injected atcell burdens ten times that necessary for tumor production byvirus-transformed cells or by transformed cells expressing an activatedras oncogene. These results suggest that the SFME cell lines may beadvantageously used as a host cell for expression of desired biologicalsubstances.

EXAMPLE IV

Effects of Serum-Free Medium Supplement Components on the Growth of SFMECells

Examination of the growth response of the SFME line in serum-free mediumindicated that the line was extremely sensitive to the omission ofinsulin or transferrin from the medium. The Swiss SFME cell line showedsignificant but less marked reduction in growth upon omission of HDL orfibronectin. Cells plated in the absence of fibronectin grow primarilyin suspension as clumps unattached to the culture dish. Omission of PDGFproduced approximately a 10% reduction in cell number after 6 days inculture, and PDGF was routinely omitted from the serum-free formulationafter passage 20. The SFME cells were also found to be absolutelydependent on the presence of EGF in the medium for survival, and diedupon its removal from the medium (90% dead within 96 hours) (FIG. 3).The EGF requirement was also observed if MCDB 402, a formulationdeveloped for Swiss 3T3 mouse embryo cells, was used as the basalnutrient medium. Flow cytometric analysis indicated that cellsmaintained in the absence of EGF accumulated in the G1 phase of the cellcycle prior to loss of viability. Omission of other individualsupplement components resulted in decreased cell growth, but did notresult in immediate cell death. The addition of the tumor promoter12-O-tetradecanoylphorbol 13-acetate (TPA) to serum-free medium fromwhich EGF had been omitted extended the life of the culture by severaldays. Both TPA and transforming growth factor beta greatly improvedattachment and spreading of SFME cells.

                  TABLE 2                                                         ______________________________________                                        EFFECT OF OMISSION OF SUPPLEMENTARY                                           COMPONENTS OF SERUM-FREE MEDIUM FOR                                           SFME CELLS                                                                    Medium                 Cell Number                                            ______________________________________                                        Complete (all supplements)                                                                           8.6 × 10.sup.5                                   Without HDL            6.3 × 10.sup.5                                   Without Fibronectin    4.8 × 10.sup.5                                   Without Insulin        1.9 × 10.sup.5                                   Without Transferrin    1.5 × 10.sup.5                                   Without EGF            all dead                                               ______________________________________                                    

Cells were plated in serum-free medium supplemented as described belowand cell number determined four days later by counting cell suspensionsin a Coulter particle counter. Cells were plated at 10⁵ /35 mm-diameterdish. Swiss SFME cells were used for the experiment. Similar resultswere obtained with BALB/c SFME cells. Cultures were grown in mediumsupplemented with 10 ug/ml bovine insulin (Sigma), 25 ug/ml humantransferrin (Sigma), 25 ng/ml EGF (Collaborative Research), and 20 ug/mlhuman HDL (Meloy) on flasks precoated with 20 ug/ml human fibronectin(Meloy), or in media from which one of the components had beenindividually omitted. Nutrient medium was a one to one mixture ofDulbecco-modified Eagle's Medium and Ham's F12 (GIBCO) containing 10 nMsodium selenite, 1.2 g/l sodium bicarbonate, 15 mM HEPES buffer, 200IU/ml penicillin, 200 ug/ml streptomycin and 25 ug/ml ampicillin.

Triiodothyronine, hydrocortisone, and transforming growth factor-betawere inhibitory to the growth of SFME cells in serum-free medium.

Mouse embryo cells derived in serum-free medium were capable of growingto very high densities (FIG. 3). Under these conditions, the cellsformed multilayered piles and aggregates. The apparent decrease ingrowth rate of serum-free mouse embryo cultures at high cell density maybe the result of both increased cell loss due to depletion of nutrientsand accumulation of cells in G1 due to rapid depletion of EGF. Survivalof the cultures at very high cell densities was precarious because ofrapid depletion of EGF. The capability for growth while suspended in alow agar concentration, or "anchorage independence" was alsoinvestigated. At low cell densities, the SFME lines form colonies insoft agar with low frequency relative to virus-transformed cells, butform colonies with high frequency at high cell densities (10⁵⁻⁶ cmplate).

EXAMPLE V

Inhibitory Effects of Serum

Although SFME cells were capable of responding in vitro to a number ofphysiologically relevant modulators of cell growth, these cells did notgrow at an appreciable rate in calf serum- or fetal calfserum-supplemented medium under conditions by which mouse embryo cellsderived by conventional protocols could be cultured indefinitely (FIGS.3 and 4). Both calf serum and platelet-free plasma were effective atinhibiting SFME cell growth; supplementation of serum-containing mediumwith the growth-stimulatory factors used in the serum-free mediummarginally improved cell growth. Inhibition of mouse embryo cellproliferation was reversible upon replacement of serum-containing mediumwith the serum-free medium. Flow cytometric analysis indicated thatcells in serum-containing medium accumulated in the G1 phase of the cellcycle.

The growth inhibitory activity of serum or plasma was partially removedby dialysis or incubation with activated charcoal for 30 minutes at 56°C. Growth inhibition could be observed upon the addition of transforminggrowth factor beta (10 ng/ml) triiodithyronine (1 nM), thyroxine orother related compounds, hydrocortisone (100 nM), dexamethasone (100 nM)or retinoic acid (1 nM). Inhibitory effects of these compounds wereenhanced by the addition of 2-10% charcoal-treated calf plasma.Combinations of these hormones were more effective than individualadditions.

EXAMPLE VI

Expression of the Ha-ras Gene in SFME Cells

Swiss and BALB SFME cells were transfected by calcium phosphateprecipitation procedures with the moelcularly cloned human Ha-rasoncogene or the ras oncogene together with the mouse cellular myc geneunder the control of the SV40 promoter. The transfected SFME cellsacquired the ability to grow in serum-free medium from which EGF hadbeen omitted; some clones were capable of growth in serum-containingmedium, particularly when supplemented with insulin and EGF. Cells wheninjected into athymic or syngeneic mice produced undifferentiatedsatcomas of undetermined type.

The ability of SFME cells to express exogenously introduced DNA wasconfirmed by transfection of SFME cells with a plasmid containing a geneconferring resistance to neomycin, followed by isolation ofneomycin-resistent cells from the transfected cultures. When the rasoncogene was also present during the transfection at a ten-fold excessover the neomycin-resistance gene, the neomycin-resistant cells werealso capable of growth in the absence of EGF.

                  TABLE 3                                                         ______________________________________                                        GROWTH OF A CLONE OF RAS-TRANSFORMED                                          SWISS SFME                                                                    Cells per plate (S 10.sup.-5)                                                 Day after                                                                            Serum-free                                                             plating                                                                              Medium     Medium - EGF 10% Serum + EGF                                ______________________________________                                        Day 1  1.5        1.1          1.4                                            Day 2  3.1        1.9          1.9                                            Day 3  4.2        3.3          2.3                                            Day 4  7.2        5.5          3.0                                            Day 5  14         12           3.6                                            Day 6  23         23           3.7                                            ______________________________________                                    

Cells were plated in serum-free medium as described and cell numberdetermined at 24 hour intervals by counting cell suspensions in aCoulter particle counter. Cells were plated at 10⁵ /35 mm-diameter dish.Ras-transformed Swiss SFME cells were used for the experiment. Similarresults were obtained with res-transformed BALB/c SFME cells.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notto be limited except as by the appended claims.

I claim:
 1. A non-tumorigenic human cell line derived from human embryocells which exhibits a predominantly diploid karyotype and is capable ofgreater than 30 generations growth in serum-free media, said cell linebeing further capable of expressing exogenously introduced genes.
 2. Thecell line of claim 1 wherein said introduced gene is a gene encoding aprotein selected from the group consisting of tissue plasminogenactivator, factor VIII, interleukin-II, insulin, growth hormone, tumornecrosis factor, superoxide dismutase, and interferon.
 3. A method forselectively controlling the growth of a non-tumorigenic human cell linewhich exhibits a predominantly diploid karyotype and is capable ofindefinite growth in serum-free media, comprising:growing said cell linein a serum-free media; and supplementing said media with a selectedamount of a biological substance selected from the group consisting ofserum, plasma, conditioned cell culture medium, tissue extracts andpurified or partially purified components thereof.
 4. The method ofclaim 3 wherein the serum-free media includes insulin, transferrin,epidermal growth factor, high-density lipoprotein and fibronectin.
 5. Amethod for producing a protein of interest, comprising:introducing intoa non-tumorigenic human cell line derived from human embryo cells whichexhibits a predominantly diploid karyotype and is capable of greaterthan 30 generations growth in serum-free media, a gene encoding aprotein of interest; growing said cell line in a serum-free media; andisolating the protein product encoded by said gene and produced by saidcell line.
 6. A method for producing a protein of interest,comprising:introducing into a non-tumorigenic murine cell line whichexhibits a predominantly diploid karyotype and is capable of indefinitegrowth in germ-free media, a gene encoding a protein of interest;growing said cell line in a serum-free media; and isolating the proteinproduct encoded by said gene and produced by said cell line.
 7. Themethod of any one of claims 5 or 6 wherein said gene is under theregulatory control of a hormone-responsive element.
 8. The method ofclaim 7 wherein said hormone-responsive element is the MMTV promoter. 9.The method of any one of claims 5 or 6 wherein said gene is a geneencoding a protein selected from the group consisting of tissueplasminogen activator, factor VIII, interleukin-II, insulin, growthhormone, tumor necrosis factor, superoxide dismutase, and interferon.10. The method of any one of claims 5 or 6 including, after the step ofisolating, purifying the protein product.
 11. The method of any one ofclaims 5 or 6 wherein the serum-free medium includes insulin,transferrin, epidermal growth factor, high-density lipoprotein andfibronectin.